Magnetic beads have gained increasing use as a convenient separation technique for many forms of cell, nucleic acid and protein isolations and analyses. In particular, the manufacturers of clinical immunoassays utilize magnetic beads both as a solid support for antibodies specifically targeted to analytes of clinical importance, and as the separation means to isolate and detect those bound analytes. But use of magnetic beads imposes a paradox. The magnetic beads must have sufficient size - normally on the order of several microns in diameter - in order to be separated in an easily achievable magnetic field. But magnetic beads of this size diffuse only very slowly, and present limited surface area for antibody binding compared to their volume. Thus the size of the current magnetic beads limits the speed and sensitivity that can be achieved in clinical assays. Through advances in template-directed polymer synthesis and nanotechnology, a new class of smart magnetic nanoparticles can be made. These magnetic nanoparticles can change from a monodispersed small diameter particle of roughly 20 nanometers diameter to a macro- aggregate of microns diameter in response to an environmental stimulus like a temperature or pH change. By using these advanced nanomaterials, assays can be developed in which the high surface to volume ratio and the small size / high diffusion of the smart magnetic nanoparticles provides for higher sensitivity and faster binding reactions compared to current magnetic bead reagents. And then a discrete pH or temperature stimulus can cause these nanoparticle reagents to co-aggregate into a macro-aggregate of micron dimensions which can be separated by a magnetic field as easily and quickly as currently used magnetic beads. This project will develop such smart assay reagents and then demonstrate their value in a model p24 (HIV) protein immunoassay. These materials present the promise of faster, more sensitive clinical immunoassays for important biomarkers of cardiac disease, cancer, endocrine and infectious diseases. PUBLIC HEALTH RELEVANCE: There exist many examples, such as assays for thyroglobulin or troponin, where increases in the sensitivity of clinical assays has led to a richer understanding of a disease state and to better medical management of patients. There are also examples, such as intra-operative thyroid stimulating hormone assays, where a faster assay leads to decreased time under anesthesia and obvious patient benefit. This project evaluates the promise of a newly developed smart reagent to enable more sensitive and faster clinical assays, and thus to contribute to better medical care.